There is a continuing need in medical practice and research, and in analytical and diagnostic procedures for rapid and accurate determinations of chemical and biological substances which are present in various fluids, such as biological fluids. For example, the presence of proteins, hormones, drugs, viruses, microorganisms, narcotics and steroids must be determined rapidly and accurately for effective research, diagnosis and treatment.
A wide variety of analytical methods have been developed in recent decades to detect the noted substances. The methods have become highly reliable and in some instances, suitable for automation, as well as suitable for use in kit form. Most of such methods rely on what are known in the art as "specific binding reactions" between a substance to be detected (identified herein as a "specific binding ligand" or "ligand") and a corresponding "receptor" which recognizes and reacts with the ligand specifically. Most well known specific binding reactions are between immunoreactants (in "immunoassays"), such as antibodies with antigens or antibodies with haptens, but others are also known (such as avidin with biotin).
In general, immunoassays can provide a qualitative or quantitative determination (or both) of the presence or absence (or quantity) of a specific antigen, antibody or antigen-antibody complex. In one form of immunoassay known as a "competitive binding immunoassay", a labeled analog of the ligand to be determined is placed in competition with a fixed amount of an appropriate antibody which can react with both the ligand and the ligand analog. The label on the analog can be appropriately detected in its "free" or complexed (that is, reacted) form. Such detection will then tell the user how much ligand is in the sample being tested.
In an alternative immunoassay format known as a "sandwich" immunoassay or immunometric assay, the ligand is contacted with two or more receptor molecules which bind to the ligand at different epitopic sites. One receptor is typically appropriately labeled and the other is either immobilized on a solid substrate, or is capable of being immobilized thereon. The amount of ligand is directly proportional to the amount of bound complex among the ligand and the two receptors.
Immunoassays have been traditionally carried out in solution, or in test devices where fluids are removed in some fashion from the reagents participating in the assay. Dry analytical elements and their use for immunoassays are described in numerous publications, including U.S. Pat. No. 4,258,001 (Pierce et al), U.S. Pat. No. 4,670,381 (Frickey et al), WO 82/2601 (published Aug. 5, 1982), EP-A-0 051 183 (published May 12, 1982) and EP-A-0 066 648 (published Dec. 15, 1982).
Improved dry analytical elements and their use in immunoassays are described in U.S. Ser. No. 938,460 (filed Aug. 31, 1992 by Belly et al) in which enzyme labels are utilized for detection. Peroxidase is the preferred enzyme label. Such elements allow for the detection of analytes present in very low concentrations using a particular wash technique to separate unbound reactants from bound (or complexed) immunoreactants.
In the immunoassays carried out in the dry analytical elements using peroxidase as the label, the stability of the peroxidase is highly important since any change in its concentration critically affects assay sensitivity. In the assays described in U.S. Ser. No. 938,460, 4'-hydroxyacetanilide is used as an electron transfer agent to enhance the rate of signal production when the enzyme label reacts with its substrate.
While 4'-hydroxyacetanilide effectively enhances assay sensitivity, it has been observed that the stability of the peroxidase label is less than desired in dry analytical elements. There is a need to have an element which demonstrates the sensitivity provided by 4'-hydroxyacetanilide, but which has improved enzyme stability.